Mechanisms of Copying of Carcinogen-damaged DNA and RNA by Translesion Polymerases

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PROJECT SUMMARY Genetic integrity is important in the maintenance of cellular homeostasis. Chemical and physical damage is well-known with DNA but less extensively studied with RNA. Miscopying of damaged bases is known to contribute to mutations and to cancer and other diseases. A focus in this laboratory for more than two decades has been DNA polymerases and their interactions with damaged DNA. Recent studies in our lab and by others have shown that DNA polymerases sometimes incorporate ribonucleoside triphosphates (rNTPs). In this laboratory, at least two of the human (h) translesion DNA polymerases, hpol η and hpol κ, have been shown to have unexpected activities with both DNA and RNA templates, including reverse transcription, DNA priming, and transcription. These activities will be studied systematically, particularly with several common lesions known to be formed in DNA and hypothesized to also be present in RNA. It is hypothesized that specific steric and bonding features of these polymerases impart these novel properties and that these biological properties are operative in cells. Features of the proposed studies include: (i) Characterization of the activity of hpol η in transcription, reverse transcription, and DNA priming. Steady- state and pre-steady-state kinetic analysis will be done to identify catalytic specificity and rate-limiting reaction steps. A key feature of the work will be several adducts in DNA and RNA (7,8-dihydro-8-oxoG (8- oxoG), 1,N6-ethenoadenine, and (thymidine-thymidine) cyclobutane pyrimidine dimer (CPD)), including analysis of how hpol η inserts correct or incorrect bases opposite each of these in RNA templates. X-Ray crystallography will be used to define features of hpol η contributing to the observed catalytic properties. Another feature of the work is analysis of levels of individual RNA adducts. (ii) Some similar questions will be addressed with hpol κ, which has been demonstrated to have reverse transcription and DNA priming activities. hpol ι has been reported to be capable of inserting rNTPs opposite undamaged DNA, an abasic site, and 8-oxoG. The mechanism will be investigated using X-ray crystal structures of ternary complexes with native and adducted DNA templates. (iii) The biological relevance of the hpol η reactions with RNA templates and insertion of ribonucleotides into DNA will be tested by investigating ribonucleotide insertion by hpol η opposite CPD (considered the most natural substrate for hpol η) under physiological conditions including typical cellular concentrations of metals and ribo- and 2ʹ′-deoxyribo-nucleotides. The effect of ribonucleotides opposite CPD will be examined with RNase H2 and nucleotide excision repair systems, in that CPD levels have been related to the presence of ribonucleotides in DNA, which in term are linked to systemic autoimmunity. XPV fibroblast cell extracts and cells will be utilized, which differ from control cells in whether they contain hpol η. Collectively these studies will provide new insight into the mechanisms of how these important polymerases act and what their biological functions are.